Project Summary: The studies herein are inspired by the concept that tumors can no longer be perceived through enumeration of the genetic mutations within the malignancy. The tumor microenvironment contributes key signals that influence cancer progression. Our long-term goal is aimed at modulating the local environment to inhibit disease progression, and these studies are aimed at identifying paracrine signaling that may drive abnormal growth. While the composition of the surrounding tumor microenvironment has been connected to disease progression, strategies for identifying the key paracrine factors emanating from the stroma are limited. Our approach is based on the intersection of secretomics (i.e., derived from RNAseq) with large-scale measurements using a TRanscriptional Activity CEll aRray (TRACER) to quantify transcription factor (TF) and micro RNA (miRNA) activity in order to identify key factors and pathways driving the observed phenotype. Macrophages are the immune cells present at the greatest levels in a primary tumor. The components of the tumor and its environment determine the phenotype of tumor associated macrophages (TAMs). The propensity of the TAMs to promote tumor growth and metastasis, or act tumoricidal or tumorostatic, is an oncongenic cell process that has been termed immune editing. The variable behavior of TAMs as a function of the tumor properties may contribute to the differential outcomes among patients, and TAMs are being investigated for their prognostic value. Specific Aim 1 will investigate paracrine communication between cancer cells and TAMs that can impact the ability of TAMs to promote or inhibit invasive phenotypes. Initial studies will investigate the bi-directional education of macrophage/monocyte populations and the cancer cells. Subsequently, mammary epithelial cells or cancer cells will be co-cultured with macrophages of varying phenotypes (pre-infiltrating monocytes, tissue-resident macrophages, and TAMs) and investigate the evolving communication between the cells that leads to tumor-promoting or tumor-inhibiting microenvironments. Specific Aim 2 will extend the TRACER technology to the single cell level (i.e., bioluminescence microscopy) to investigate signaling within the most invasive cancer cells. Heterogeneity within the tumor population is increasingly appreciated as an important contributor to the observed cancer phenotype. These studies capture the active TFs/miRNA associated with invasion, and identify the factors with distinct activity that underlie the differential phenotype of the invasive cell relative to the non-invasive cells within the population. The ability to capture the dynamic activity of numerous TF/miRNAs at single cell resolution can be employed to track the cues driving cell fate decisions, which is not achievable through other methods and provides novel perspectives into cancer biology and cancer immunology. Finally, we investigate the paracrine signaling and TF/miRNA activity in the context of chemotherapy and impact on the tumor microenvironment, as compensatory signaling may serve as a therapeutic target to enhance the efficacy of current cancer treatments.